Richard E. Roberts

Role of the extracellular signal-regulated kinase (Erk) signal transduction cascade in α2 adrenoceptor-mediated vasoconstriction in porcine palmar lateral vein

The mechanism of α2 adrenoceptor‐mediated vasoconstriction is unknown, but may involve activation of voltage‐sensitive calcium channels, and/or a protein tyrosine kinase. Recently the extracellular signal‐regulated kinase (Erk) cascade, often an event downstream of tyrosine kinase activation, has been shown to mediate vasoconstriction to a variety of agents. The aim of this present study was to determine the involvement of the Erk signal transduction cascade in α2 adrenoceptor‐mediated vasoconstriction, and to confirm the involvement of activation of voltage‐sensitive calcium channels, and protein tyrosine kinase. Contractions to the α2 adrenoceptor agonist UK14304 in the porcine palmar lateral vein in vitro were reduced 70 – 80% by the MEK inhibitors PD98059 (10 – 50 μM) and U0126 (10 – 50 μM), indicating the involvement of the Erk signal transduction cascade. Immunoblots also demonstrated an increase in the phosphorylated (activated) form of Erk in palmar lateral vein segments after contraction with UK14304, which was inhibited by PD98059 and U0126. The calcium channel blockers nifedipine and verapamil, or removal of extracellular calcium inhibited UK14304‐induced contractions and phosphorylation of Erk, demonstrating the importance of an influx of extracellular calcium. UK14304‐induced contractions were inhibited by PP2 (1 – 10 μM), a selective inhibitor of Src tyrosine kinases, but not by PP3, an inactive analogue. PP2 also prevented the phosphorylation of Erk by UK14304. These data demonstrate that α2 adrenoceptor‐mediated vasoconstriction in the porcine palmar lateral vein is dependent upon activation of the Erk signal transduction cascade, which is downstream of an influx of extracellular calcium, and activation of Src tyrosine kinases.

Hydrogen sulphide-induced relaxation of porcine peripheral bronchioles.

Hydrogen sulphide (H2S) is an endogenous gasotransmitter. Although it has been shown to elicit responses in vascular and other smooth muscle preparations, a role for endogenously produced H2S in mediating airway tone has yet to be demonstrated. Therefore, the aim of this study was to determine whether H2S is produced within the airways and to determine the functional effect on airway tone.

α2‐Adrenoceptor‐mediated contractions of the porcine isolated ear artery: evidence for a cyclic AMP‐dependent and a cyclic AMP‐independent mechanism

1 The aim of this study was to determine the conditions under which the α2‐adrenoceptor agonist UK14304 produces vasoconstriction in the porcine isolated ear artery. 2 UK14304 (0.3 μm) produced a small contraction of porcine isolated ear arteries which was 7.8±3.3% of the response to 60 mm KC1. Similar sized contractions were obtained after precontraction with either 30 nm angiotensin II, or 0.1 μm U46619 (8.2±1.8% and 10.2±2.6% of 60 mm KC1 response, respectively). However, an enhanced α2‐adrenoceptor response was uncovered if the tissue was precontracted with U46619, and relaxed back to baseline with 1–2 μm forskolin before the addition of UK14304 (46.9±9.6% of 60 mm KC1 response). 3 The enhanced responses to UK14304 in the presence of U46619 and forskolin were not inhibited by the α1‐adrenoceptor antagonist prazosin (0.1 μm), but were inhibited by the α2‐adrenoceptor antagonist rauwolscine (1 μm), indicating that the enhanced responses were mediated via postjunctional α2‐adrenoceptors. 4 In the presence of 0.1 μm U46619 and 1 mm isobutylmethylxanthine (IBMX), 1 μm forskolin produced an increase in [3H]‐cyclic AMP levels in porcine isolated ear arteries. Addition of 0.3 μm UK14304 prevented this increase. 5 The enhanced UK14304 response was dependent upon the agent used to relax the tissue. After relaxation of ear arteries precontracted with 10 nm U46619 and relaxed with forskolin the UK14304 response was 46.9±9.6% of the 60 mm KC1 response, and after relaxation with sodium nitroprusside (SNP) the response was 24.8±3.3%. However, after relaxation of the tissue with levcromakalim the UK14304 response was only 8.2±1.7%, which was not different from the control response in the same tissues (12.2±5.6%). An enhanced contraction was also obtained after relaxation of the tissue with the cyclic AMP analogue dibutyryl cyclic AMP (23.2±1.3%) indicating that at least part of the enhanced response to UK14304 is independent of the ability of the agonist to inhibit cyclic AMP production. 6 Relaxation of U46619 contracted ear arteries with SNP could be inhibited by the NO‐sensitive guanylyl‐cyclase inhibitor 1H‐[1,2,4] oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ) indicating that production of cyclic GMP is necessary for the relaxant effect of SNP. However, ODQ had no effect on the relaxation of tissue by forskolin, suggesting that this compound does not act via production of cyclic GMP. Biochemical studies showed that while forskolin increases the levels of cyclic AMP in the tissues, SNP had no effect on the levels of this cyclic nucleotide. 7 In conclusion, enhanced contractions to the α2‐adrenoceptor agonist UK14304 can be uncovered in porcine isolated ear arteries by precontracting the tissue with U46619, followed by relaxation back to baseline with forskolin, SNP or dibutyryl cyclic AMP before addition of UK14304. There was a greater contractile response to UK14304 after relaxation with forskolin than with SNP or dibutyryl cyclic AMP, suggesting that cyclic AMP‐dependent and‐ independent mechanisms are involved in the enhancement of the UK14304 response.

Enhancement of α2-adrenoceptor-mediated vasoconstriction by the thromboxane-mimetic u46619 in the porcine isolated ear artery: role of the ERK-MAP kinase signal transduction cascade

α2‐Adrenoceptor‐mediated contractions in porcine blood vessels can be enhanced in the presence of the thromboxane‐mimetic U46619, and forskolin. The aim of this study was to determine the role of U46619 in the enhanced contractions, and to determine whether signalling through the ERK–MAP kinase pathway is involved. Responses to the α2‐adrenoceptor agonist UK14304 (1 μM) were increased from 22±3% of the response to 60 mM KCl to 68±12% (n=8, mean±s.e.m.) in the presence of a low concentration of U46619 (<20% of the 60 mM KCl response). Both the direct and the U46619‐enhanced UK14304 responses were inhibited by 50 μM PD98059, an inhibitor of the ERK–MAP kinase pathway. UK14304‐induced contractions were associated with an increase in ERK2 phosphorylation, indicating an increased activity. In the presence of U46619, there was an enhanced phosphorylation of ERK2. U46619 on its own had no effect on ERK phosphorylation. Both the direct and enhanced UK14304 contractions were inhibited in the absence of extracellular calcium. These conditions also prevented the increase in ERK2 phosphorylation. This indicates a role for calcium influx in the enhanced contractions. In conclusion, this study demonstrates that precontraction with the thromboxane‐mimetic U46619 enhances α2‐adrenoceptor‐mediated vasoconstriction through the enhancement of the ERK–MAP kinase pathway, and influx of extracellular calcium.

Sex differences in endothelial function in porcine coronary arteries: a role for H2O2 and gap junctions?

Cardiovascular risk is higher in men and postmenopausal women compared with premenopausal women. This may be due to sex differences in endothelial function. Here, sex differences in endothelial function of porcine coronary arteries (PCAs) were investigated.

Altered GAP-43 Immunoreactivity in Regenerating Sciatic Nerve of Diabetic Rats

Experimental diabetes in the rat is associated with impaired axon regeneration. Successful regeneration depends on the construction of axonal growth cones and establishment of appropriate target connections. The growth-associated protein (GAP)–43 is a major component of the axonal growth cone, and its synthesis and axonal transport are markedly increased during regeneration. The purpose of this study was to determine the effect of experimental diabetes on the synthesis and axonal transport of GAP–43 in regenerating sciatic nerves. Rats were rendered diabetic with 50 mg/kg streptozotocin i.p. Four weeks later, the rats were anesthetized, and one sciatic nerve was crushed to induce regeneration. After 2 weeks, nerves were ligated, and 6 h later, nerve pieces proximal to the ligature and dorsal root ganglia were removed, and proteins were separated by PAGE. Western blots of gels were probed with antibody 10E8/E7 against GAP-43. The presence of GAP-43 was confirmed by immunohistochemistry of nerve sections. Densitometric analysis of the blots showed a 45% reduction in native GAP-43 immunoreactivity in nerve pieces proximal to the ligature (P < 0.05; n = 7). Northern blots of total RNA extracted from pooled dorsal root ganglia were probed with a 32P-radiolabeled cDNA probe for GAP-43. There was no significant difference in the amount of GAP-43 mRNA between diabetic and nondiabetic rats. Immunohistochemistry of sciatic nerve confirmed the reduction in GAP-43 immunoreactivity. We conclude that a defect in turnover or axonal transport of GAP-43 may contribute to the impaired peripheral nerve regeneration in diabetes.

Sex differences in the role of NADPH oxidases in endothelium-dependent vasorelaxation in porcine isolated coronary arteries

The present study examined whether vascular function, expression and activity of NADPH oxidases differ between sexes in porcine isolated coronary arteries (PCAs) using selective Nox inhibitors, ML-171 and VAS2870. Vascular responses of distal PCAs were examined under myographic conditions in the presence of a range of inhibitors. Nox activity in PCA homogenates was assessed using lucigenin-enhanced chemiluminescence. Protein expression of Nox1, Nox2 and Nox4 was compared using Western immunoblotting. The presence of ML-171 or DPI had no effect on the bradykinin-induced vasorelaxation in PCAs from females. In males, DPI shifted the EC50 2.8-fold to the right. In the presence of L-NAME and indomethacin, DPI and ML-171 had no effect in females, but enhanced the bradykinin-induced vasorelaxation in males. ML-171 had no effect on the forskolin-induced vasorelaxation but decreased the potency of U46619-induced tone in both sexes in the absence or presence of endothelium. VAS2870 had no effect on the bradykinin-induced vasorelaxation in both sexes but reduces the EDH-type response in males only. Nox activity was reduced by DPI and ML-171, but not VAS2870 in PCAs from both sexes. Protein expression of Nox1 and Nox2 in PCAs was higher in males compared to females whereas Nox4 was higher in females. Inhibition of Nox with ML-171 enhances while VAS2870 reduces the EDH-type response in PCAs from males but not females. This indicates that Nox-generated ROS play a role in the EDH-type response in males with differences attributed to the differential expression of Nox isoforms. This may underlie the greater oxidative stress observed in males.

Targeted inhibition of Gq signaling induces airway relaxation in mouse models of asthma

Local pharmacological inhibition of Gq results in airway relaxation in mouse models of asthma. Breathing freely Obstructive lung diseases are common disorders characterized by airway narrowing. Because some patients do not respond well to current therapies or suffer from side effects, new drugs are needed. Matthey et al. now report that the selective Gq inhibitor FR900359 reduces airway tone in mouse, pig, and human airways ex vivo and decreases airway resistance in mouse models of asthma in vivo. The compound has the advantage that it can be locally applied to the lung via inhalation and shows promising properties that may prove useful for treating obstructive airway disease. Obstructive lung diseases are common causes of disability and death worldwide. A hallmark feature is aberrant activation of Gq protein–dependent signaling cascades. Currently, drugs targeting single G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptors (GPCRs) are used to reduce airway tone. However, therapeutic efficacy is often limited, because various GPCRs contribute to bronchoconstriction, and chronic exposure to receptor-activating medications results in desensitization. We therefore hypothesized that pharmacological Gq inhibition could serve as a central mechanism to achieve efficient therapeutic bronchorelaxation. We found that the compound FR900359 (FR), a membrane-permeable inhibitor of Gq, was effective in silencing Gq signaling in murine and human airway smooth muscle cells. Moreover, FR both prevented bronchoconstrictor responses and triggered sustained airway relaxation in mouse, pig, and human airway tissue ex vivo. Inhalation of FR in healthy wild-type mice resulted in high local concentrations of the compound in the lungs and prevented airway constriction without acute effects on blood pressure and heart rate. FR administration also protected against airway hyperreactivity in murine models of allergen sensitization using ovalbumin and house dust mite as allergens. Our findings establish FR as a selective Gq inhibitor when applied locally to the airways of mice in vivo and suggest that pharmacological blockade of Gq proteins may be a useful therapeutic strategy to achieve bronchorelaxation in asthmatic lung disease.

α2‐Adrenoceptor and NPY receptor‐mediated contractions of porcine isolated blood vessels: evidence for involvement of the vascular endothelium

Enhanced contractions to the α2‐adrenoceptor agonist UK14304 and neuropeptide Y (NPY) in the porcine ear artery can be uncovered by pharmacological manipulation. The aim of this study was to determine whether similar pharmacological manipulation can uncover enhanced contractions in the porcine splenic artery, and to determine whether the endothelium modulates these responses. UK14304 (0.3 μM) and NPY (0.1 μM) produced small contractions of the porcine splenic artery. After pre‐contraction of the tissue with U46619, followed by relaxation with forskolin, the responses to both UK14304 and NPY were enhanced. Enhanced contractions to both UK14304 and NPY were also obtained after relaxation with SNP. These results demonstrate that, as in the porcine ear artery, α2‐adrenoceptors and NPY receptors are able to produce enhanced contractile responses through both adenylyl cyclase‐dependent and ‐independent signal transduction pathways. Removal of the endothelium had no significant effect on responses to UK14304 either alone or in the presence of U46619 and forskolin in the porcine splenic artery. On the other hand, responses to UK14304 after relaxation with SNP were reduced after endothelium‐denudation in both the porcine splenic artery and ear artery. Similar results were obtained with NPY in the porcine ear artery. In conclusion, enhanced contractile responses to UK14304 and NPY in the porcine splenic artery can be uncovered using methods similar to those employed in the porcine ear artery. Under certain conditions the responses to both agents are modulated by the endothelium. These data highlight further the similarities in the signal transduction pathways used by both α2‐adrenoceptors and NPY receptors to induce vasoconstriction.

α2 Adrenoceptor-mediated vasoconstriction in porcine palmar lateral vein: role of phosphatidylinositol 3-kinase and EGF receptor transactivation

α2 Adrenoceptors cause vasoconstriction in the porcine palmar lateral vein through a mechanism involving the ERK signal transduction cascade, calcium influx, and a Src tyrosine kinase. The aim of the present study was to determine if phosphatidylinositol 3‐kinase (PI 3‐kinase) and/or epidermal growth factor (EGF) receptor transactivation are also involved. α2 Adrenoceptor‐mediated vasoconstriction and ERK2 activation in the porcine palmar lateral vein was inhibited in the presence of either the PI 3‐kinase inhibitor LY294002, or the EGF receptor tyrosine kinase inhibitor AG1478 suggesting the involvement of both PI 3‐kinase and EGF receptor transactivation. Akt phosphorylation was increased in segments of porcine palmar lateral vein contracted with UK14304 indicating an increase in Akt activation. This is a further indication that PI 3‐kinase is involved in α2 adrenoceptor‐mediated vasoconstriction. Akt activation was inhibited by the Src tyrosine kinase inhibitor PP2, and removal of extracellular calcium. UK14304 (10 μM) stimulated an increase in intracellular calcium in segments of palmar lateral vein. This was inhibited by removal of extracellular calcium, but not by nifedipine suggesting the rise in calcium is due to influx of calcium through non‐L type calcium channels. The increase in calcium was also inhibited by LY294002 indicating that PI 3‐kinase is upstream of calcium influx. These data indicate that α2 adrenoceptor‐mediated vasoconstriction in the porcine palmar lateral vein is dependent upon stimulation of PI 3‐kinase, leading to an influx of calcium. This results in activation of the EGF receptor tyrosine kinase, and finally activation of ERK–MAP kinase.